Processes for making inoculum cultures used to produce edible mushrooms on a suitably prepared substrate are well known. Processes suitable for the efficient large-scale production of mycelia inoculum, however, are notably absent from the art.
Early production methods involved the use of a solid culture medium such as tobacco, grain and manure which have been shown to support the growth of vegetative mycelia. These methods require an undesireably extended period of time (i.e., 30 to 60 days) for the mycelium to attain a suitable developmental stage, and are labor-intensive, requiring several manual transfers of aseptic materials as production is scaled up. The manual transfers have traditionally used very low ratios of inoculum to new substrate to maximize yield of product. In addition, efforts to produce commercial amounts of mycelia inoculum using a solid culture medium process occasionally resulted in adverse selection pressure of a particular mushroom strain.
A submerged fermentation process using liquid media reduces the time required to produce mushroom mycelia inoculum. Some techniques involving the use of liquid ("submerged") culture techniques are taught in the prior art. For example, U.S. Pat. No. 3,286,399 discloses the production of mushroom spawn after sowing with mycelium or spores in a submerged culture that consists of carbohydrate- and nitrogen-containing nutrients. U.S. Pat. No. 5,077,201 discloses the production of blue indigo pigment from a strain of morel mushroom by submerged fermentation in a nutrient medium containing a carbon and a nitrogen substrate. U.S. Pat. No. 4,977,902 discloses the production of Pleurotus sp. and Volvaria sp. in a liquid media adapted for plastic packaging.
These submerged culture techniques, however, are not suitable for producing large amounts of mycelia suitable for commercial mushroom production. Submerged culture basidiomycete mycelia by conventional fermentation techniques forms very large, clumped colonies that are dense balls of mycelia. This cultural problem limits the amount of biomass that can be produced and forms the same selection pressures on the biomass that cold be expected in solid state culture. Large colony formation limits the points of inoculation that could be obtained from the fermentation broth. Accordingly, a submerged fermentation process that can use liquid media for the generation of larger amounts of mycelia suitable for commercial mushroom production is needed.
The suitability of submerged culture techniques to produce liquid mushroom mycelia inoculum for commercial use also depends on the efficiency of submerged mycelial growth. Growth efficiency depends largely on the content of medium nutrients and the growth conditions of the fermentation process. For example, Agaricus sp. does not grow well on starch and corn gluten in the absence of sufficient amounts of soluble protein and other nutrients, such as those suggested by U.S. Pat. No. 3,286,399. European Pat. No. 284,421 discloses the cultivation of filamentous fungi by inoculation onto a substrate comprising a variety of nutrients ranging from carbohydrates, nitrogens, lipids, nucleotides, sterols, vitamins and inorganic compounds to plant and bacterial extracts. This medium is sufficient to "sustain" the growth of the fungi, but does not "enhance" the growth of mycelia. U.S. Pat. No. 4,512,103 describes mushroom growth on undefined liquid nutrients produced by thermophilic digestion of biodegradable organic materials.
Prior art references provide only limited descriptions of processes in which mushroom growth is enhanced by the selection of a specified nutrient media. U.S. Pat. No. 4,370,159 discloses a 30% increase in growth and yield of edible mushroom by use of a nutrient particle comprising a matrix of denatured protein containing droplets of fat and active protein. This medium is useful for "mature mycelial" in commercial compost which is near the cropping stage. It is not suitable for non-mature mycelia and spores. U.S. Pat. No. 4,420,319 discloses nutritional enhancement additives comprising an agglomerate of activator and slow-release nutrient particles. Although this material reduces the time required for mushroom spawn to reach fruition and further retards premature aging of the cells, it appears to be suitable primarily for mushroom spawn which has already been inoculated onto seed grain. U.S. Pat. No. 4,818,268 discloses an osmoprotectant for enhancing mushroom growth which comprises carrier particles having water-soluble phosphoglyceride material attached. This osmoprotectant is used for enhancing and prolonging mushroom growth and for extending cropping by protecting the later flushes against the effects of increased osmotic stress. None of these prior art references, however, are directed to a medium for enhancing the growth of mycelia suitable for inoculation at a commercial mushroom production level. Identification of a liquid medium capable of enhancing the growth of mushroom mycelia to a commercially acceptable level is needed.
In addition to the identification of nutrients for use in liquid media to enhance the growth of mycelial inoculum, a commercially feasible process must be capable also of continuous or semi-continuous preparation of inoculated substrate in an automated, aseptic system. To achieve this commercial goal, a medium is needed that is capable of producing large quantities of log-phase inocula at a uniform growth stage. At present, only low yields of Agaricus mycelia from submerged culture growth have been achieved. Accordingly, a liquid medium suitable for the production of large amounts of log-phase mycelial inoculum at a uniform growth stage is needed.
The prior art further is silent with respect to a continuous or semi-continuous method for inoculating sterile substrates useful in the commercial production of mushrooms. Commercially feasible mushroom spawn must be capable of producing a cost-effective, aseptic fermentation of submerged culture mycelia that can be coupled with a continuous or semi-continuous inoculation of sterile substrates. These substrates must be compatible with current methods of inoculation at commercial mushroom farms which include the use of grains such as rye or millet as a substrate. These grains are convenient to admix into the compost. Additionally, the process should provide for the direct inoculation of the compost with the liquid culture through conventional spawning equipment fitted with suitable liquid discharge equipment.